Choreography of a proton pump : Studies of charge-transfer reactions in cytochrome c oxidase

Sammanfattning: In the last step of cellular respiration, electrons from metabolites are transferred to molecular oxygen, mediated by the enzyme complexes of the respiratory chain. Some of these enzyme complexes couple these redox reactions to formation of an electrochemical proton gradient across the cell membrane. The proton gradient is used e.g. by ATP synthase to drive synthesis of ATP. The terminal enzyme complex in the respiratory chain, cytochrome c oxidase (CytcO), catalyses reduction of O2 to water. In this process it contributes to maintaining the electrochemical proton gradient by two separate mechanisms: (i) by uptake of electrons and protons from the opposite sides of the membrane (for O2 reduction to water). (ii) by proton pumping across the membrane. Protons used in the O2 reduction, as well as protons that are pumped, are taken up through two different proton-uptake pathways, the D and the K pathways. In addition, a third proton-transfer pathway has been suggested for the mitochondrial CytcOs, namely the H pathway. So far, the molecular mechanism by which CytcO pumps protons has not been determined. In this work we have studied proton- and electron-transfer reactions in aa3-type CytcOs, with the aim of understanding the functional design of the proton-pumping machinery in CytcO. First, we studied structural variants of CytcO from the bacterium Rhodobacter (R.) sphaeroides, where an amino-acid at position 425, previously shown to undergo redox-induced conformational changes, was substituted. The results point to a link between redox-induced structural changes and intramolecular proton-transfer rates through the D pathway. Second, we studied the electron distribution in the “activated” oxidized (OH) state of CytcO, by using an electrostatic complex of CytcO and cytochrome c. We also investigated electron-transfer reactions linked to proton pumping in structural variants of CytcO from R. sphaeroides and the yeast Saccharomyces (S.) cerevisiae, with mutations in the proposed D and H proton-uptake pathways. The data indicate that the S. cerevisiae mitochondrial CytcO uses the D pathway for proton uptake and pumping as the R. sphaeroides CytcO. Lastly, we studied reactions linked to proton uptake and pumping in structural variants of CytcO from R. sphaeroides with alterations in both proton-uptake pathways. The data elucidated the mechanism of proton transfer and gating in CytcO.